PEX5 Research Articles

Lifelong course of Zellweger spectrum disorders: A cohort study

Objective: Zellweger spectrum disorders (ZSD) are a group of genetic metabolic disorders caused by a defect in peroxisome biogenesis due to mutations in one of the PEX genes. Although originally seen as a rapidly lethal pediatric disease, an increasing number of patients with a relatively mild or aspecific phenotype were recognized over the past few years as a result of improved clinical and biochemical characterization. Consequently, a substantial percentage of ZSD patients reach adulthood nowadays. Since only limited data on the natural course of the disease in these relatively mild patients are available, this retrospective cohort study aims to gain insight in the natural course of these patients. Methods: Clinical and laboratory data were collected retrospectively from a large cohort of patients with a genetically confirmed ZSD. All patients were regularly followed up and examined as a part of standard patient care. Results: A total of 22 patients in this ZSD cohort reached adulthood. All patients, except one patient with PEX10 mutations, had vision and hearing impairment. Neurological symptoms comprised pyramidal tract dysfunction, cerebellar syndrome and peripheral neuropathy, in most cases eventually leading to a gait disorder. Two patients suffered from epileptic seizures. In multiple adult patients, a relatively rapid progression of mainly neurological symptoms is described after a long period of stabilization, accompanied by diffuse white matter abnormalities and general cerebral atrophy on MRI- imaging. Conclusion: Most patients with a ZSD at the mild end of the spectrum suffer from slowly progressive neurological symptoms, vision and hearing impairment. Rapid progression of neurological symptoms after a long period of stabilization can occur, whilst biochemical abnormalities have normalized over the years. The exact pathophysiology of this rapid deterioration remains to be elucidated.

RNA silencing of PEX6 gene causes decrease in pigmentation, sporulation and pathogenicity of Fusarium oxysporum

Peroxisomes are single membrane‐bound organelles that play a pivotal role in various developmental processes in all eukaryotic cells. This study targeted the PEX6 gene, which encodes for peroxisomal biogenesis factor 6, by RNA interference (RNAi) in Fusarium oxysporum f. sp. lycopersici. Fusarium oxysporum is a soilborne filamentous, hemibiotrophic fungus that invades tomato roots and colonizes the xylem vessels, thereby causing complete wilting of infected tomato plants. The expression of FoPEX6 in F. oxysporum was found to be higher during early stages of growth and development. The FoPEX6 gene was isolated and a hairpin RNAi construct was prepared and introduced into F. oxysporum 4471 through glass‐bead transformation. The fungal transformation status, i.e. integration, expression and presence of the intended small interfering RNAs (siRNAs), was confirmed by PCR, qPCR and stem‐loop PCR, respectively. The silenced fungal transformants exhibited reduced pigmentation and a significant reduction in sporulation as compared to the wild type. They also showed dramatic reduction in pathogenicity (virulence) on tomato, based on root infection and fruit invasion assays. These results suggest that PEX6 has a central role in pigmentation, sporulation and pathogenicity in F. oxysporum.

A novel PEX1 mutation in a Moroccan family with Zellweger spectrum disorders

Mutations in the PEX1 gene are usually associated with recessive inherited diseases including Zellweger spectrum disorders. In this work, we identified a new pathogenic missense homozygous PEX1 mutation (p.Leu1026Pro, c.3077T>C) in two Moroccan syndromic deaf siblings from consanguineous parents. This variation is located in the P-loop containing nucleoside triphosphate hydrolase of protein domain and probably causes an alteration in the hydrolysis of ATP.

Biochemical and clinical profiles of 52 Tunisian patients affected by Zellweger syndrome

Zellweger syndrome (ZS) is a peroxisome biogenesis disorder attributed to a mutation of the PEX genes family. The incidence of this disease in Africa and the Arab world remains unknown. This contribution is aimed at describing the clinical phenotype and biochemical features in Tunisian patients with ZS in order to improve the detection and management of this severe disorder. A total of 52 patients diagnosed with ZS and 60 age- and sex-matched healthy controls were included in this study. Patients were recruited during the past 21 years, and the diagnosis of ZS was based on clinical and biochemical characteristics. Plasma very long chain fatty acids (VLCFA) were analyzed using capillary gas chromatography. The estimated incidence of ZS was calculated using the Hardy-Weinberg formula. The estimated incidence of ZS is 1/15,898 in Tunisia. Age at diagnosis varied between 3 days and 18 months. Severe neurological syndrome, polymalformative features, and hepatodigestive signs were observed in 100%, 67.9%, and 32% of patients, respectively. Values for plasma C26:0 and C26:0/C22:0 and C24:0/C22:0 ratios were noticeably higher in ZS patients than in controls. Distributions of values were completely different for C26:0 (0.10-0.37 vs. 0.001-0.009), C26:0/C22:0 ratio (0.11-1.29 vs. 0.003-0.090), and C24:0/C22:0 ratio (1.03-3.18 vs. 0.4-0.90) in ZS patients versus controls, respectively. This study highlights the high incidence of ZS in Tunisia and the possibility of simple and reliable biochemical diagnosis, thus permitting early genetic counseling for families at risk.

Attenuation of Neuronal Lipid Accumulations and Delayed Myelination in the Brain of Postnatal PEX2 Zellweger Mice

Zellweger syndrome is a peroxisomal biogenesis disorder, characterized by the absence of peroxisomes. Peroxin (PEX) genes, which regulate the import process of peroxisome matrix proteins, are mutated in patients with Zellweger syndrome. The majority of peroxisomal reactions involve lipids, including fatty acid beta-oxidation and ether phospholipid biosynthesis. However, in the absence of peroxisomes, these metabolic pathways are severely disrupted, resulting in toxic accumulations of very long chain fatty acids and many other metabolic disturbances (Wanders and Romeijn, 1998). Previous research on mice with deletion of the PEX2 gene (PEX2-/-) revealed an absence of normal peroxisomes, central nervous system malformations with an abnormality of neuronal migration and neuronal lipid accumulation in embryonic and newborn brain (Faust, 2003; Faust and Hatten, 1997; Faust et al., 2001). Using the PEX2-/- mouse model, we characterized the distribution and time course of abnormal neuronal lipid accumulation in the postnatal mutant brain. Oil red staining in PEX2 mutant mice illustrates persistence of abnormal lipid deposits in postnatal day 3 (P3) brain but their near complete disappearance in P12-P36 PEX2 mutants, with the exceptions of olfactory bulb and ependymal lining. This loss of neuronal lipid accumulation suggests these lipids may be removed by alternate non-peroxisomal mechanisms in the developing brain. Alternatively, changing metabolic requirements in developing versus mature neurons may differentially affect lipid metabolism in the absence of peroxisomes. Lastly, our results show that myelin sheaths are visibly attenuated in the brains of PEX2 mutant mice at P15. This attenuation persists somewhat through P36. The relationship of these myelin abnormalities to specific peroxisomal lipid dysfunctions remains to be investigated.

Pex35 is a regulator of peroxisome abundance.

Peroxisomes are cellular organelles with vital functions in lipid, amino acid and redox metabolism. The cellular formation and dynamics of peroxisomes are governed by PEX genes; however, the regulation of peroxisome abundance is still poorly understood. Here, we use a high-content microscopy screen in Saccharomyces cerevisiae to identify new regulators of peroxisome size and abundance. Our screen led to the identification of a previously uncharacterized gene, which we term PEX35, which affects peroxisome abundance. PEX35 encodes a peroxisomal membrane protein, a remote homolog to several curvature-generating human proteins. We systematically characterized the genetic and physical interactome as well as the metabolome of mutants in PEX35, and we found that Pex35 functionally interacts with the vesicle-budding-inducer Arf1. Our results highlight the functional interaction between peroxisomes and the secretory pathway.

Generation of Peroxisome-Deficient Somatic Animal Cell Mutants.

Cell mutants with a genetic defect affecting various cellular phenotypes are widely utilized as a powerful tool in genetic, biochemical, and cell biological research. More than a dozen complementation groups of animal somatic mutant cells defective in peroxisome biogenesis have been successfully isolated in Chinese hamster ovary (CHO) cells and used as a model system reflecting fatal human severe genetic disorders named peroxisome biogenesis disorders (PBD). Isolation and characterization of peroxisome-deficient CHO cell mutants has allowed the identification of PEX genes and the gene products peroxins, which directly leads to the accomplishment of isolation of pathogenic genes responsible for human PBDs, as well as elucidation of their functional roles in peroxisome biogenesis. Here, we describe the procedure to isolate peroxisome-deficient mammalian cell mutants from CHO cells, by making use of an effective, photo-sensitized selection method.

Pex14/17, a filamentous fungus-specific peroxin, is required for the import of peroxisomal matrix proteins and full virulence of Magnaporthe oryzae.

Peroxisomes are ubiquitous organelles in eukaryotic cells that fulfil a variety of biochemical functions. The biogenesis of peroxisomes requires a variety of proteins, named peroxins, which are encoded by PEX genes. Pex14/17 is a putative recently identified peroxin, specifically present in filamentous fungal species. Its function in peroxisomal biogenesis is still obscure and its roles in fungal pathogenicity have not yet been documented. Here, we demonstrate the contributions of Pex14/17 in the rice blast fungus Magnaporthe oryzae (Mopex14/17) to peroxisomal biogenesis and fungal pathogenicity by targeting gene replacement strategies. Mopex14/17 has properties of both Pex14 and Pex17 with regard to its protein sequence. Mopex14/17 is distributed at the peroxisomal membrane and is essential for efficient peroxisomal targeting of proteins containing peroxisomal targeting signal 1. MoPEX19 deletion leads to the cytoplasmic distribution of Mopex14/17, indicating that the peroxisomal import of Pex14/17 is dependent on Pex19. The knockout mutants of MoPEX14/17 show reduced fatty acid utilization, reactive oxygen species (ROS) degradation and cell wall integrity. Moreover, Δmopex14/17 mutants show delayed conidial generation and appressorial formation, and a reduction in appressorial turgor accumulation and penetration ability in host plants. These defects result in a significant reduction in the virulence of the mutant. These data indicate that MoPEX14/17 plays a crucial role in peroxisome biogenesis and contributes to fungal development and pathogenicity.

Severe early onset retinitis pigmentosa in a Moroccan patient with Heimler syndrome due to novel homozygous mutation of PEX1 gene

Heimler syndrome (HS) is a rare recessive disorder characterized by sensorineural hearing loss (SNHL), amelogenesis imperfecta, nail abnormalities, and occasional or late-onset retinal pigmentation. It is the mildest form known to date of peroxisome biogenesis disorder caused by hypomorphic mutations of PEX1 and PEX6 genes. We report on a second Moroccan family with Heimler syndrome with early onset, severe visual impairment and important phenotypic overlap with Usher syndrome. The patient carried a novel homozygous missense variant c.3140T > C (p.Leu1047Pro) of PEX1 gene. As standard biochemical screening of blood for evidence of a peroxisomal disorder did not provide a diagnosis in the individuals with HS, patients with SNHL and retinal pigmentation should have mutation analysis of PEX1 and PEX6 genes.

Experimental study of PEX gene transfection into rat C6 glioma cells mediated by SonoVue combined with ultrasonic irradiation

Objective To investigate the feasibility of pEGFP-C1-PEX transfection into rat C6 glioma cells mediated by SonoVue combined with ultrasonic irradiation, and observe the effect of PEX gene on C6 glioma cells. Methods The C6 cells were cultured in vitro and divided into 6 groups: control group, ultrasound+ microbubbles group, plasmid+ microbubbles group, plasmid+ ultrasound group, plasmid+ ultrasound+ microbubbles group. Twenty-four hours after appropriate treatment, the transfection efficiency was assessed by fluorescent microscopy, the mRNA expression of PEX was detected by RT-PCR, and the cell cycle change was assessed by flow cytometry. Results The transfection efficiency of PEX gene was highest, the specific PEX electrophoresis strips was seen, and the percentage of G0/G1 phase rised to (75.09±2.46)%, S period cells decreased to (17.91±2.63)% in plasmid+ ultrasound+ microbubbles group, compared with other groups, the differences were statistically significant (all P<0.05). Conclusions SonoVue and ultrasonic irradiation can enhance the transfection of PEX gene into rat C6 glioma cells; PEX gene can inhibit the proliferation of rat C6 glioma cells by affecting the cell cycle in vitro. Key words: Ultrasonography; Microbubbles; Glioma; PEX gene

Diagnostic and prognostic value of in vivo proton MR spectroscopy for Zellweger syndrome spectrum patients.

Defects in the biogenesis of peroxisomes cause a clinically and genetically heterogeneous group of neurometabolic disorders, the Zellweger syndrome spectrum (ZSS). Diagnosis predominantly is based on characteristic clinical symptoms, a typical biochemical profile, as well as on identification of the molecular defect in any of the 12 known human PEX genes. The diagnostic workup can be hindered if the typical clinical symptoms are missing and predicting the clinical course of a given patient is almost unfeasible. As a safe and noninvasive method to analyze specific chemical compounds in localized brain regions, in vivo proton magnetic resonance spectroscopy (MRS) can provide an indication in this diagnostic process and may help predict the clinical course. However, to date, there are very few reports on this topic. In this study, we performed localized in vivo proton MRS without confounding contributions from T1- and T2-relaxation effects at 2 Tesla in a comparably large group of seven ZSS patients. Patients' absolute metabolite concentrations in cortical gray matter, white matter, and basal ganglia were assessed and compared with age-matched control values. Our results confirm and extend knowledge about in vivo MRS findings in ZSS patients. Besides affirmation of nonspecific reduction of N-acetylaspartate + N-acetylaspartylglutamate (tNAA) in combination with lipid accumulation as a diagnostic hint for this disease group, the amount of tNAA loss seems to reflect disease burden and may prove to be of prognostic value regarding the clinical course of an already diagnosed patient.

Cholic acid therapy in Zellweger spectrum disorders.

IntroductionZellweger spectrum disorders (ZSDs) are characterized by a failure in peroxisome formation, caused by autosomal recessive mutations in different PEX genes. At least some of the progressive and irreversible clinical abnormalities in patients with a ZSD, particularly liver dysfunction, are likely caused by the accumulation of toxic bile acid intermediates. We investigated whether cholic acid supplementation can suppress bile acid synthesis, reduce accumulation of toxic bile acid intermediates and improve liver function in these patients.MethodsAn open label, pretest-posttest design study was conducted including 19 patients with a ZSD. Participants were followed longitudinally during a period of 2.5 years prior to the start of the intervention. Subsequently, all patients received oral cholic acid and were followed during 9 months of treatment. Bile acids, peroxisomal metabolites, liver function and liver stiffness were measured at baseline and 4, 12 and 36 weeks after start of cholic acid treatment.ResultsDuring cholic acid treatment, bile acid synthesis decreased in the majority of patients. Reduced levels of bile acid intermediates were found in plasma and excretion of bile acid intermediates in urine was diminished. In patients with advanced liver disease (n = 4), cholic acid treatment resulted in increased levels of plasma transaminases, bilirubin and cholic acid with only a minor reduction in bile acid intermediates.ConclusionsOral cholic acid therapy can be used in the majority of patients with a ZSD, leading to at least partial suppression of bile acid synthesis. However, caution is needed in patients with advanced liver disease due to possible hepatotoxic effects.Electronic supplementary materialThe online version of this article (doi:10.1007/s10545-016-9962-9) contains supplementary material, which is available to authorized users.

Correlation of peroxisome pathway reactive oxygen species oxidative stress gene and its correlation with the antitumor sensitivity of artesunate against pancreatic cancer

Objective To explore the screening of peroxisome pathway reactive oxygen species (ROS) oxidative stress gene and its correlation with the antitumor sensitivity of artesunate against pancreatic cancer. Methods Based on microarray mRNA expressions of 55 tumor cell lines in the National Cancer Institute common database, peroxisome pathway-related key genes which were significant correlation with half-inhibitory concentration (IC50) values of artesunate antitumor activity against human pancreatic cancer were selected by Kendall test. The candidate genes associated with artesunate sensitivity were identified and their mRNA expressions in pancreatic cancer cells were tested using fluorescent quantitative PCR. The contents of peroxidase in pancreatic cancer cells were detected through the DAB staining. Results Thirteen key genes mRNA expressions in peroxidase pathways were significantly correlated with IC50 values for artesunate antitumor activity. Compared with normal liver cells HL-7702 (1.00), CRAT (2.89±0.06), PEX11B (1.90±0.07) and PEX16 (1.35±0.07) mRNA expression levels were significantly increased in pancreatic cancer Panc-1 cells which sensitive to artesunate (t=33.00, P<0.01; t=17.85, P<0.01; t=4.54, P<0.05). While CAT (1.43±0.03), SOD1 (2.07±0.04) and SOD2 (1.15±0.01) mRNA expression levels were also significantly increased in Panc-1 cells which sensitive to artesunate (t=11.71, P<0.01; t=35.85, P<0.01; t=13.22, P<0.01). However, PEX12 (0.51±0.02), CAT (0.47±0.02), PRDX1 (0.43±0.01), and SOD1 (0.44±0.01) mRNA expression levels in pancreatic cancer BXPC-3 cells which resistant to artesunate were significantly lower than that of HL-7702 cells (t=37.53, P<0.01; t=16.52, P<0.01; t=84.20, P<0.01; t=48.24, P<0.01). DAB staining showed that the positive expression rate of peroxisomal content was apparently higher in Panc-1 cells (61.5%) than that of HL-7702 cells (43.8%), with a significant difference (χ2=16.11, P<0.01). Conclusion Peroxisome and its related ROS antioxidant enzymes CAT, PRDX1, SOD gene expression may be the important factors that affect artesunate antitumor activity against human pancreatic cancer. Key words: Pancreatic neoplasms; Peroxisomes; Reactive oxygen species; Antineoplastic agents; Artesunate

Novel PEX3 Gene Mutations Resulting in a Moderate Zellweger Spectrum Disorder.

Peroxisome biogenesis disorders (PBDs) may have a variable clinical expression, ranging from severe, lethal to mild phenotypes with progressive evolution. PBDs are autosomal recessive disorders caused by mutations in PEX genes, which encode proteins called peroxins, involved in the assembly of the peroxisome. Patient Description: We herein report a patient who is currently 9years old and who is compound heterozygous for two novel mutations in the PEX3 gene. Mild biochemical abnormalities of the peroxisomal parameters suggested a Zellweger spectrum defect in the patient. Sequence analysis of the PEX3 gene identified two novel heterozygous, pathogenic mutations. Mutations in PEX3 usually result in a severe, early lethal phenotype. We report a patient compound heterozygous for two novel mutations in the PEX3 gene, who is less affected than previously reported patients with a defect in the PEX3 gene. Our findings indicate that PEX3 defects may cause a disease spectrum similar as previously observed for other PEX gene defects.

Dysmorphic Facial Features and Other Clinical Characteristics in Two Patients with PEX1 Gene Mutations.

Peroxisomal disorders are a group of genetically heterogeneous metabolic diseases related to dysfunction of peroxisomes. Dysmorphic features, neurological abnormalities, and hepatic dysfunction can be presenting signs of peroxisomal disorders. Here we presented dysmorphic facial features and other clinical characteristics in two patients with PEX1 gene mutation. Follow-up periods were 3.5 years and 1 year in the patients. Case I was one-year-old girl that presented with neurodevelopmental delay, hepatomegaly, bilateral hearing loss, and visual problems. Ophthalmologic examination suggested septooptic dysplasia. Cranial magnetic resonance imaging (MRI) showed nonspecific gliosis at subcortical and periventricular deep white matter. Case II was 2.5-year-old girl referred for investigation of global developmental delay and elevated liver enzymes. Ophthalmologic examination findings were consistent with bilateral nystagmus and retinitis pigmentosa. Cranial MRI was normal. Dysmorphic facial features including broad nasal root, low set ears, downward slanting eyes, downward slanting eyebrows, and epichantal folds were common findings in two patients. Molecular genetic analysis indicated homozygous novel IVS1-2A>G mutation in Case I and homozygous p.G843D (c.2528G>A) mutation in Case II in the PEX1 gene. Clinical findings and developmental prognosis vary in PEX1 gene mutation. Kabuki-like phenotype associated with liver pathology may indicate Zellweger spectrum disorders (ZSD).

A Novel Mutation of the PEX16 Gene in a Patient with Slowly Progressive Zellweger Syndrome

Zellweger syndrome (ZS) disorders are autosomal recessive peroxisomal biogenesis diseases mainly characterized by neonatal onset severe neurodevelopmental delay, profound hypotonia, craniofacial dysmorphism, hepatic dysfunction, polyneuropathy and loss of hearing and vision. There is a wide genetic heterogeneity that while most ZS disorders are rapidly progressive and incurable, and patients rarely survive through their first birthday, many patients have late-onset and mild ZS phenotypes. Human PEX16 is an integral membrane protein first isolated by Honsho and plays a central role in peroxisomal membrane biogenesis. According to the few existing reports, the severity and the natural course of PEX16-mutated patients are unclear, and therapy has not been discussed. Herein and based on existing research, we report and discuss the case of a young female diagnosed with slowly progressive ZS involving a novel PEX16 mutation.

Peroxisome biogenesis and human peroxisome-deficiency disorders.

Peroxisome is a single-membrane-bounded ubiquitous organelle containing a hundred different enzymes that catalyze various metabolic pathways such as β-oxidation of very long-chain fatty acids and synthesis of plasmalogens. To investigate peroxisome biogenesis and human peroxisome biogenesis disorders (PBDs) including Zellweger syndrome, more than a dozen different complementation groups of Chinese hamster ovary (CHO) cell mutants impaired in peroxisome biogenesis are isolated as a model experimental system. By taking advantage of rapid functional complementation assay of the CHO cell mutants, successful cloning of PEX genes encoding peroxins required for peroxisome assembly invaluably contributed to the accomplishment of cloning of pathogenic genes responsible for PBDs. Peroxins are divided into three groups: 1) peroxins including Pex3p, Pex16p and Pex19p, are responsible for peroxisome membrane biogenesis via Pex19p- and Pex3p-dependent class I and Pex19p- and Pex16p-dependent class II pathways; 2) peroxins that function in matrix protein import; 3) those such as Pex11pβ are involved in peroxisome division where DLP1, Mff, and Fis1 coordinately function.

Peroxisome biogenesis disorders in the Zellweger spectrum: An overview of current diagnosis, clinical manifestations, and treatment guidelines.

Peroxisome biogenesis disorders in the Zellweger spectrum (PBD-ZSD) are a heterogeneous group of genetic disorders caused by mutations in PEX genes responsible for normal peroxisome assembly and functions. As a result of impaired peroxisomal activities, individuals with PBD-ZSD can manifest a complex spectrum of clinical phenotypes that typically result in shortened life spans. The extreme variability in disease manifestation ranging from onset of profound neurologic symptoms in newborns to progressive degenerative disease in adults presents practical challenges in disease diagnosis and medical management. Recent advances in biochemical methods for newborn screening and genetic testing have provided unprecedented opportunities for identifying patients at the earliest possible time and defining the molecular bases for their diseases. Here, we provide an overview of current clinical approaches for the diagnosis of PBD-ZSD and provide broad guidelines for the treatment of disease in its wide variety of forms. Although we anticipate future progress in the development of more effective targeted interventions, the current guidelines are meant to provide a starting point for the management of these complex conditions in the context of personalized health care.

Zellweger spectrum disorders: clinical overview and management approach.

Zellweger spectrum disorders (ZSDs) represent the major subgroup within the peroxisomal biogenesis disorders caused by defects in PEX genes. The Zellweger spectrum is a clinical and biochemical continuum which can roughly be divided into three clinical phenotypes. Patients can present in the neonatal period with severe symptoms or later in life during adolescence or adulthood with only minor features. A defect of functional peroxisomes results in several metabolic abnormalities, which in most cases can be detected in blood and urine. There is currently no curative therapy, but supportive care is available. This review focuses on the management of patients with a ZSD and provides recommendations for supportive therapeutic options for all those involved in the care for ZSD patients.

Role of AAA+-proteins in peroxisome biogenesis and function

Mutations in the PEX1 gene, which encodes a protein required for peroxisome biogenesis, are the most common cause of the Zellweger spectrum diseases. The recognition that Pex1p shares a conserved ATP-binding domain with p97 and NSF led to the discovery of the extended family of AAA+-type ATPases. So far, four AAA+-type ATPases are related to peroxisome function. Pex6p functions together with Pex1p in peroxisome biogenesis, ATAD1/Msp1p plays a role in membrane protein targeting and a member of the Lon-family of proteases is associated with peroxisomal quality control. This review summarizes the current knowledge on the AAA+-proteins involved in peroxisome biogenesis and function. This article is part of a Special Issue entitled: Peroxisomes edited by Ralf Erdmann.